Primary maternal human cytomegalovirus (HCMV) infection affects 1-3% of pregnancies and causes birth defects in 25% of congenitally infected babies. The placentas are damaged from inflammation, edema, hypoxia, and fibrosis. HCMV replicates in decidual (maternal) cells, placental (fetal) cytotrophoblasts (CTB) and blood vessels, and the chorionic villi become progressively edematous reducing blood perfusion. Accumulation of fluid in the placental-fetal unit also suggests lymphatic vessels in the decidua have diminished capacity to remove excess fluid. How lymphatic functions could be impaired by direct infection or paracrine effects in utero is not known. Preliminary studies showed that HCMV replicates in lymphatic endothelial cells (LEC) and dysregulates differentiation and migration in vitro. Importantly, infected lymphatic vessels were found in the decidua of congenitally-infected placentas. To study the molecular mechanisms in vivo, we developed a novel model of human placentation in which xenografts of human placental villi (fetal) and decidua (maternal) are implanted in severe-combined immunodeficient (SCID-hu) mice. Fetal CTB differentiate and induce lymphangiogenesis, and new lymphatic vessels form like those in the human decidua. We showed that a pathogenic HCMV strain replicates in CTB of placental villi and decidual implants and that lymphangiogenesis was impaired. Vascular endothelial growth factors A and C (VEGF-A/C) upregulated in infected CTB could have paracrine effects on LEC functions in vivo. Our overarching hypothesis is that HCMV impairs lymphangiogenesis by (i) direct infection of decidual lymphatic vessels and (ii) paracrine effects of growth factors and cytokines secreted from placental CTB (fetal) and decidual cells (maternal) in the pregnant uterus. The specific aims are as follows. Aim 1. Study direct HCMV infection of decidual LEC to identify molecular pathways that dysregulate integrins that function as downstream effectors of chemotaxis, migration and cord formation. Aim 2. Study paracrine effects of VEGF-related proteins from HCMV-infected placental CTB (fetal). Examine factors that dysregulate lymphangiogenesis in placental villous xenografts in SCID-hu mice. Aim 3. Investigate cytokines from infected decidual fibroblasts (maternal) that affect lymphangiogenesis. Study paracrine factors in xenografts of decidual tissue infected with HCMV in explants and in SCID-hu mice. Confirmation of our hypothesis will be directly relevant to clinical applications of new treatments to prevent placental edema during pregnancy using antibodies or small molecules that block paracrine factors and inflammatory cytokines. These could have clinical application in conjunction with biotherapeutic antibodies that suppress HCMV replication, prevent placental dysfunction and improve chances for a healthy baby. PUBLIC HEALTH RELEVANCE: Identifying the molecular mechanisms that impair functions of infected lymphatic endothelial cells, and secreted factors from infected placental (fetal) and decidual (maternal) cells, could lead to new clinical interventions to prevent placental edema and associated pregnancy complications from congenital infection.